This research project is concerned with the structure and mechanism of action of the active sites of delta super 5-3-ketosteroid isomerase from P. testosteroni and the development of new techniques for the investigation of ligand-protein interactions. Highlights of progress during the past year (6-72 - 6-73) include: 1. Successful affinity labelling of the isomerase using photoexcited 3-ketosteroids as active- site-directed reagents. 2. Discovery that a major reaction which occurs during 3- ketosteroid dependent photoinactivation involves chemical modification of a phenylalanine residue of the enzyme. The chemical nature of this modification is under investigation. 3. A minor, but significant, reaction during photoinactivation involves covalent attachment of the steroid to the protein; this also is being further investigated. 4. An intermediate which occurs in primary amine catalyzed isomerization of delta super 5-3-ketosteroid to delta super 4- 3-ketosteroids can be reductively trapped by sodium borohydride. Isolation and characterization of the reduced intermediate has been accomplished. Its properties are consistent with its being derived from a Schiff's base intermediate. 5. Development of an affinity chromatographic purification of isomerase using a Sepharose column containing covalently bound testosterone.